Antiretroviral Drug Metabolism in Humanized PXR-CAR-CYP3A-NOG Mice

Abstract

Antiretroviral drug (ARV) metabolism is linked largely to hepatic cytochrome P450 activity. One ARV drug class known to be metabolized by intestinal and hepatic CYP3A are the protease inhibitors (PIs). Plasma drug concentrations are boosted by CYP3A inhibitors such as cobisistat and ritonavir (RTV). Studies of such drug-drug interactions are limited since the enzyme pathways are human specific. While immune-deficient mice reconstituted with human cells are an excellent model to study ARVs during human immunodeficiency virus type 1 (HIV-1) infection, they cannot reflect human drug metabolism. Thus, we created a mouse strain with the human pregnane X receptor, constitutive androstane receptor, and CYP3A4/7 genes on a NOD.Cg-Prkdc^scid Il2rg^tm1Sug /JicTac background (hCYP3A-NOG) and used them to evaluate the impact of human CYP3A metabolism on ARV pharmacokinetics. In proof-of-concept studies we used nanoformulated atazanavir (nanoATV) with or without RTV. NOG and hCYP3A-NOG mice were treated weekly with 50 mg/kg nanoATV alone or boosted with nanoformulated ritonavir (nanoATV/r). Plasma was collected weekly and liver was collected at 28 days post-treatment. Plasma and liver atazanavir (ATV) concentrations in nanoATV/r-treated hCYP3A-NOG mice were 2- to 4-fold higher than in replicate NOG mice. RTV enhanced plasma and liver ATV concentrations 3-fold in hCYP3A-NOG mice and 1.7-fold in NOG mice. The results indicate that human CYP3A-mediated drug metabolism is reduced compared with mouse and that RTV differentially affects human gene activity. These differences can affect responses to PIs in humanized mouse models of HIV-1 infection. Importantly, hCYP3A-NOG mice reconstituted with human immune cells can be used for bench-to-bedside translation.

Fig. 1.

Experimental timeline for nanoformulated drug treatment and blood and tissue collection. NanoATV/r or nanoATV were administered on days 0, 3, 7, 14, and 21 by intramuscular injection to hCYP3A-NOG and NOG mice. Whole blood was collected into heparinized tubes on days 1, 3, 7, 14, 21, and 28 and plasma was prepared. Liver tissue was collected at day 28.

Fig. 2.

Drug plasma concentrations in nanoATV or nanoATV/r-treated mice. (A) ATV plasma concentrations in humanized hCYP3A-NOG (hCYP3A) or NOG mice treated intramuscularly with 20 or 50 mg/kg nanoATV/r or 50 mg/kg nanoATV on days 0, 3, 7, 14, and 21. (B) RTV plasma concentrations in hCYP3A and NOG mice treated with 20 or 50 mg/kg nanoATV/r. Data are expressed as mean ± S.E.M. N = 5–8.

Fig. 3.

Sex-dependent differences in plasma drug concentrations in mice treated with nanoATV/r. ATV (A) and RTV (B) plasma concentrations over time in humanized hCYP3A-NOG (hCYP3A) and NOG male and female mice treated intramuscularly with 50 mg/kg nanoATV/r on days 0, 3, 7, 14, and 21. Data are expressed as mean ± S.E.M. N = 3 (female) or 4 (male).

Fig. 4.

Liver drug concentrations in hCYP3A-NOG (hCYP3A) and NOG mice. Mice were treated intramuscularly with 20 or 50 mg/kg nanoATV/r or 50 mg/kg nanoATV on days 0, 3, 7, 14, and 21 and livers were collected on day 28. Liver ATV (A) and RTV (B) concentrations were determined by UPLC tandem MS. Data are expressed as mean ± S.E.M. *Significantly different than similarly treated NOG mice (P < 0.05). ^†Significantly different than hCYP3A mice treated with 50 mg/kg nanoATV/r (P < 0.05).

Fig. 5.

Sex-dependent differences in liver drug concentrations in mice treated with nanoATV/r. Mice were treated intramuscularly with 20 or 50 mg/kg nanoATV/r or 50 mg/kg nanoATV on days 0, 3, 7, 14, and 21 and livers were collected on day 28. Liver ATV (A) and RTV (B) concentrations were determined by UPLC tandem MS. Males, solid bars; females, hatched bars. Data are expressed as mean ± S.E.M. *Significantly different than respectively treated male mice (P < 0.05). N = 4 to 5 (nanoATV/r-treated males); N = 3 (nanoATV/r-treated females; nanoATV-treated males); N = 2 (nanoATV-treated females).

Fig. 6.

Liver CYP3A4, PXR, and CAR mRNA levels in mice treated with nanoATV or nanoATV/r. Expression of human and mouse genes for CYP3A4, PXR, and CAR in liver were determined by real-time PCR using human (hCYP3A) and mouse (NOG) specific primers in mice treated with 50 mg/kg nanoATV or 50 mg/kg nanoATV/r. Significant differences were determined by one-tailed Students t test at *P < 0.05; **P < 0.01. N = 4 to 5 (nanoATV/r-treated males); N = 3 (nanoATV/r-treated females; nanoATV-treated males); N = 2 (nanoATV-treated females).